At present there is no generally applicable cure for HIV infections of humans. While some development of anti-viral drugs and therapies exists, these have not yet proven capable of eliminating the virus from the infected patient. This is in large part due to the establishment of infected tissue reservoirs for the virus in which the virus may remain relatively quiescent for long periods of time, continuously shedding small amounts of virus into the patient to maintain or reestablish infection. A need continues to exist in the Art to cure a patient of the HIV infection.
While highly active anti-retroviral therapy (HAART) provides some suppression of HIV infection and allows restoration of immune function, effectively circumventing the decline into AIDS, HAART treatment alone cannot provide a cure for the infection. In addition, there remain several problems with this approach, including the expense of long term treatments, the continuing contagiousness of the patient, development of escape mutations, and toxicity associated with long term drug treatments. Discontinuation of HAART allows rapid rebound of the infection. Similarly, rebound of infection may occur through evolution and selection of quasi and mutated species resistant to HAART. Both of these viral rebounds result from an inability to completely clear the virus from infected patients due to the presence of persistently infected, long-lived lymphocyte or macrophage cell populations that serve as reservoirs for the virus (see Blankson et al., 2002).
Alternative approaches utilizing transplantation of ex vivo genetically transformed lymphocyte and macrophage lineage cells or progenitor stem cell populations are being explored as a means of suppressing HIV infection in susceptible cell populations (See Rossi et al., 2007). One major drawback to many of these approaches is their focus on suppressing the production of new virus in re-implanted cells while still allowing these cells to be re-infected. This approach allows the re-establishment and perpetuation of the very reservoirs that permit the evolution of quasi species of the virus, and allows escape mutants with these mutations to expand.
The first confirmed catalytic RNA, the cis-splicing intron of T. thermophila pre-rRNA, termed ‘ribozyme’ to describe the RIBO nucleic acid-based enzyme, was reported by Kruger et al. (1982). This intron excised itself from the highly purified mature rRNA in a solution of magnesium and guanosine in a cell-free system. Later, this intron was configured to splice together RNA on two separate molecules by two successive trans-esterification reactions (Sullenger & Chec, 1994).
The trans-splicing Group I intron reaction targets an RNA molecule through the use of antisense guide sequences that hybridize with the target RNA and permit cleavage at a specific uracil, releasing the downstream sequence (see FIG. 1). This step frees a 3′ OH downstream of the uracil cleavage point to act as a nucleophile which carried out the covalent joining of the upstream target RNA fragment to an intron-associated 3′ exon, resulting in a new, continuous RNA molecule. This reaction can be designed to generate a new contiguous open reading frame, leading to a transcript molecule that encodes a protein product that is present if and only if splicing has occurred.
In a trans-splicing reaction, two separate segments of the intron are utilized to specify the RNA sequence the ribozyme will target. The internal guide sequence and external guide sequence (IGS and EGS, respectively) are each complementary to a segment of the target. The IGS is limited in size to roughly 9 base pairs near the target uracil, and forms what is termed the P1 helix with the target, where the reaction will eventually occur. The EGS can be of nearly any length and forms a transient helix downstream of the target uracil. A longer EGS will increase the specificity and affinity of the intron towards its target RNA (Kohler et al., 1999).
The trans-splicing reaction is catalyzed by the P10 helix that is formed by the 3′ end of the intron in the vicinity of the splicing reaction to guide the 3′ exon to the proper ligation point. This step is vital to the second step of the reaction as it enables the free 3′ OH of the cleavable uracil to attack the phosphate backbone upstream of the 3′ exon, allowing covalent joining of the 3′ exon to the upstream cleavage product to create a new, seamless mRNA suitable for translation.
Group I intron trans-splicing has been used in a number of applications including repair of mutant B-globin mRNA (Byun et al., 2003), restoration of wild-type p53 activity in three cancerous cell lines (Lander et al., 2001), re-establishment of the function of the canine skeletal muscle chloride channel (Waterson et al., 2002), and induction of p16 activity in a pancreatic cell line (Kastanos et al., 2004). The trans-splicing group I intron has proven to be an effective anti-cancer therapy in model systems. Researchers were able to cause the cell-specific death of human colon cancer cells by targeting an mRNA coding for the carcinoembryonic antigen utilizing HSV-tk as a 3′ exon followed by ganciclovir treatment (Jung & Lee, 2006). This same group, using similar methods, achieved group I intron catalyzed trans-splicing of the liver-cancer upregulated α-fetoprotein (AFP) in human liver cancer cells (Won & Lee, 2007) and the mouse homologue of the cancer associated cytoskeleton-associated protein 2 in mammalian cells (Kim et al., 2007). Also reported is the cell-specific cytotoxicity induced via generation of diphtheria toxin A (DTA), or ganciclovir/herpes simplex V thymidine kinase (HSV-tk)-induced apoptosis in cells expressing the tumo. These Group 1 trans-splicing introns r associated hTERT subunit of telomerase (Jung et al., 2005; Kwon et al., 2005), and trans-splicing of the hepatitis C virus internal ribosome entry site (HCV-IRES) (Ryu et al., 2003).
Despite these and other reports, a need continues to exist in the medical arts for more effective and long-lasting treatments for human immunodeficiency virus (HIV) infection, and in halting the progression of the infection to AIDS.